The present invention is related generally to immunoassays for detecting hepatitis C virus and specifically to methods of detecting hepatitis C virus in biological samples, methods of screening blood products for hepatitis C virus, and kits therefor.
Hepatitis C virus (HCV) was first identified and characterized as the primary cause of post transfusion non-A, non-B hepatitis (NANBH) by Houghton, et al. In addition to providing substantial information concerning HCV, Houghton, et al., and their collaborators have disclosed a number of general and specific immunological reagents and methods. See, e.g. Houghton, et al., EPO Pub. No. 318,216; Houghton, et al., EPO Pub. No. 388, 232; Choo, et al., Science, 1989, 244, 359-362; Kuo, et al., Science, 1989, 244, 362-364; Takeuchi, et al., J. Gen. Virol., 1990, 71, 3027-3033; Takeuchi, et al., Gene, 1990, 91, 287-291; Takeuchi, et al., Nucl. Acids Res., 1990, 18, 4626; Miyamura, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 983-987: Saito, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 6547-6549; Choo, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 2451-2455; Han, et al., Proc. Natl. Acad. Sci. USA, 88, 1711-1715; Houghton, et al., Hepatology, 1991, 14, 381-388; and Weiner, et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 3468-3472. These publications provide the art with an extensive background on HCV generally, as well as the manufacture and uses of HCV polypeptide immunological reagents. For brevity, therefore, the disclosure of these publications in particular are incorporated herein by reference in their entirety.
Others have readily applied and extended the work of Houghton, et al. See, e.g. Highfield, et al., UK Pat App. 2,239,245 (The Welcome Foundation Ltd.); Wang, EPO Pub. No. 442,394 (United Biomedical Inc.); Leung, et al., EPO Pub. No. 445,423 (Abbott Laboratories); Habits, et al., EPO Pub. No. 451,891 (Akzo N.V.); Reyes, et al., PCT Pub No. WO 91/15516 (Genelabs Inc.); Maki, et al., EPO Pub. No. 468,657 (Tonen Corp.); and Kamada, et al., EPO Pub. No. 469,348 (Shionogi Seiyaku K.K). See also, Matsuura, et al., J. Virology, 1992, 66, 1425; Kato, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 9524-9528; Takamizawa, et al., J. Virol., 1991, 65, 1105-1113; Chiba, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 4641-4645; Harada, et al., J. Virol., 1990, 65, 3015-3021; Hijikata, et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 5547-5551; Okamoto, et al., Jpn. J. Exp. Med, 1990, 60, 167-177; Yuasa, et al., J. Gen. Virol., 1991, 72, 2021-2024; and Watanabe, et al., Int. J. Cancer, 1991, 48, 340-343.
Sensitive, specific methods for screening and identifying carriers of HCV and HCV-contaminated blood or blood products, as well as monitoring patients undergoing treatment, are important advances in medicine. Post-transfusion hepatitis (PTH) occurs in approximately 10% of transfused patients, and HCV has accounted for up to 90% of these cases. The major problem in this disease is the frequent progression to chronic liver damage (25-55%) relative to other hepatitises, such as type B.
Patient care as well as the prevention of transmission of HCV by blood and blood products or by close personal contact require reliable diagnostic and prognostic tools, such as, for example, HCV antibodies, to detect proteins related to HCV infection. Such antibodies are also useful as agents for monitoring treatment regimens for patients with HCV. Since HCV is a relatively new agent, a continuing need exists to define additional immunological reagents that will allow further study of the clinical course of disease and the epidemiology of HCV in the population.
Current methodology for detecting HCV focuses on detecting HCV-specific antibodies. See, for example, Hada, et al., Acta Med. Okayama, 1992, 46, 365-70; Miyamura, et al., EPO Pub. No. 0537626; Lok, et al., Hepatology, 1993, 18, 497-502; Wang, et al., Vox Sang, 1992, 62, 21-4; Kleinman, et al., Transfusion, 1992, 32, 805-813; Leon, et al., Vox Sang, 1996, 70, 213-16; Lesniewski, et al., J. Med. Virol., 1995, 45, 415-22; and Inoue, et al., J. Gen. Virol., 1992, 73, 2151-54. A major disadvantage to detecting antibodies which react with HCV is that seroconversion has already occurred and the patient may already have a well-established viral infection. Alternatively, if an individual is determined to be HCV antibody reactive, it may simply mean that the individual has been exposed to HCV sometime in the past and may not be presently infected.
Other methods for detecting HCV comprise using PCR. See, for example, Francois, et al., J. Clin. Microbiol., 1993, 31, 1189-93. HCV envelope proteins have also been detected by immunohistochemical analysis of hepatocytes in patients with chronic liver disease. However, these assays do not easily lend themselves to a clinical setting. Hiramatsu, et al., Hepatology, 1992, 16, 306-311.
Other methods also are directed to detecting HCV core protein. See, for example, Orito, et al., Gut, 1996, 39, 876-80and Kashiwakuma, et al., J. Immunol. Methods, 1996, 190, 79-89. These methods include a protein-capture fluorescence enzyme immunoassay (FEIA), a traditional sandwich ELISA assay, using monoclonal antibodies reactive with recombinant HCV core protein. The method consists of using one monoclonal antibody coated on a solid phase as a capture antibody and beta-D-galactosidase conjugated monoclonal antibody as the antigen detection signal antibody. These assays, however, require very tedious sample preparation procedures including, for example, polyethyleneglycol precipitation, NaOH denaturation, retritration of the sample to neutral pH and addition of Triton X-100 to the sample preparation prior to starting the assay. Such assays are not convenient in the clinical setting. Indeed, what is required is an easy, fast immunoassay for detection of HCV antigens.
Applicants have developed an immunoassay system which can detect HCV envelope antigens, E1 and E2, without tedious sample preparation procedures. Applicants"" invention provides a means to detect free HCV antigens prior to seroconversion, antigen disappearance after acute infection, and/or interferon therapy and, thus, is useful as a diagnostic and for blood screening and evaluating the effectiveness of drug treatment. Thus, Applicants"" invention is a significant improvement for detecting HCV in biological samples.
The present invention is directed to methods for detecting hepatitis C virus in biological samples comprising contacting the sample with an anti-human antibody and at least one monoclonal anti-hepatitis C virus envelope protein antibody under conditions that allow an immunologic reaction between the antibodies and the sample, and detecting the presence of immune complexes of the antibodies and the envelope protein.
The present invention is also directed to methods for detecting hepatitis C virus in a biological sample comprising contacting an anti-human antibody attached to a solid phase with a polyclonal anti-hepatitis C virus envelope protein antibody, contacting the sample to the polyclonal antibody, contacting the sample with at least one detectably-labeled, monoclonal anti-hepatitis C virus envelope protein antibody under conditions that allow an immunologic reaction between the antibodies and the sample, and detecting the presence of immune complexes of the antibodies and the envelope protein and/or the presence of free envelope protein.
The present invention is also directed to methods of screening blood components or blood for hepatitis C virus prior to the use of such blood or blood component to prepare blood products comprising reacting a body component from a potential donor with an anti-human antibody and at least one monoclonal anti-hepatitis C virus envelope protein antibody under conditions that allow an immunologic reaction between the antibodies and the body component, detecting the presence of immune complexes formed between the antibodies and hepatitis C virus envelope proteins, and discarding any blood or blood component from the donor if the complexes are detected.
The present invention is also directed to kits for detecting hepatitis C virus in a biological sample comprising an anti-human antibody, at least one monoclonal anti-hepatitis C virus envelope protein antibody, control standards, and instructions for use of the kit components.